Comparative analysis of Listeria monocytogenes plasmid transcriptomes reveals common and plasmid‐specific gene expression patterns and high expression of noncoding RNAs

Abstract Recent research demonstrated that some Listeria monocytogenes plasmids contribute to stress survival. However, only a few studies have analyzed gene expression patterns of L. monocytogenes plasmids. In this study, we identified four previously published stress‐response‐associated transcriptomic data sets which studied plasmid‐harboring L. monocytogenes strains but did not include an analysis of the plasmid transcriptomes. The four transcriptome data sets encompass three distinct plasmids from three different L. monocytogenes strains. Differential gene expression analysis of these plasmids revealed that the number of differentially expressed (DE) L. monocytogenes plasmid genes ranged from 30 to 45 with log2 fold changes of −2.2 to 6.8, depending on the plasmid. Genes often found to be DE included the cadmium resistance genes cadA and cadC, a gene encoding a putative NADH peroxidase, the putative ultraviolet resistance gene uvrX, and several uncharacterized noncoding RNAs (ncRNAs). Plasmid‐encoded ncRNAs were consistently among the highest expressed genes. In addition, one of the data sets utilized the same experimental conditions for two different strains harboring distinct plasmids. We found that the gene expression patterns of these two L. monocytogenes plasmids were highly divergent despite the identical treatments. These data suggest plasmid‐specific gene expression responses to environmental stimuli and differential plasmid regulation mechanisms between L. monocytogenes strains. Our findings further our understanding of the dynamic expression of L. monocytogenes plasmid‐encoded genes in diverse environmental conditions and highlight the need to expand the study of L. monocytogenes plasmid genes' functions.

In addition to well-characterized chromosomally encoded stress response systems, some L. monocytogenes plasmids increase tolerance to various stress conditions, including elevated temperature, ultraviolet light, salt concentrations, lactic acid, and disinfectants Elhanafi et al., 2010;Naditz et al., 2019;Pontinen et al., 2017). On average, 47%−54% of L. monocytogenes strains harbor a putative plasmid, although plasmid prevalence between different sequence types is highly variable (Chmielowska et al., 2021;Schmitz-Esser et al., 2021). L. monocytogenes plasmids are highly conserved across strains in a modular fashion in which certain regions possess high degrees of sequence similarity to regions on other plasmids (Kuenne et al., 2010;Schmitz-Esser et al., 2021).
However, while modules themselves can be highly conserved, the number and identity of modules on a given L. monocytogenes plasmid can be highly variable (Kuenne et al., 2010;Schmitz-Esser et al., 2021).
Whole transcriptome sequencing is widely used in L. monocytogenes research to elucidate genes of interest involved in virulence, saprophytic stress survival, and gene expression regulatory mechanisms Assisi et al., 2021;Behrens et al., 2014;Cortes et al., 2020;Guariglia-Oropeza et al., 2018;Kragh & Truelstrup Hansen, 2019;Marinho et al., 2019;Mraheil et al., 2011;Soni et al., 2011;Tang et al., 2015;Vivant et al., 2017;Wehner et al., 2014;Wurtzel et al., 2012). However, transcriptome sequencing studies that include an analysis of plasmid gene expression in L. monocytogenes are limited in number Cortes et al., 2020;Kragh & Truelstrup Hansen, 2019). Several studies that conducted gene expression analysis on plasmid-harboring L. monocytogenes strains did not evaluate plasmid gene expression due to methodological constraints (Assisi et al., 2021;Guariglia-Oropeza et al., 2018;Tang et al., 2015). In these studies, the transcriptome sequencing reads were mapped to a reference L. monocytogenes genome different than the strain used for the gene expression experiments. These reference strains (L. monocytogenes EDG-e and 10403S) do not contain a plasmid, and as a result, all plasmid reads were discarded in the read mapping. We thus sought to utilize these available, novel transcriptomic data to improve our understanding of the role of L. monocytogenes plasmids in different conditions. These data sets cover three genetically distinct plasmids: the well-studied L. monocytogenes plasmid pLM80 and two novel putative plasmids, pLM5446 and pLM7802, from the whole-genome shotgun sequences of L. monocytogenes isolates FSL  and FSL R8-7802 (CU-259-322), respectively.

| Review of the literature
This study aimed to improve our understanding of L. monocytogenes plasmid gene expression based on existing published data. Therefore, we searched NCBI PubMed for studies based on the following criteria: (1) transcriptome sequencing must have been performed on an L. monocytogenes strain; (2) the L. monocytogenes strain used in the transcriptomics experiment must have a sequenced and assembled genome; (3) the strain must harbor a putative plasmid; (4) the study must not have reported plasmid gene expression data. In strains where the existence of a plasmid was unknown, the genomes were subjected to protein BLAST (BLASTp) searches (method described below) with known L. monocytogenes plasmid replication protein RepA protein sequences as a query, and annotations from significant hits of the query sequences were manually reviewed for known L. monocytogenes plasmid proteins.
After a detailed literature search, we found three studies that fulfilled all criteria (Tables 1 and 2) (Assisi et al., 2021;Guariglia-Oropeza et al., 2018;Tang et al., 2015). Two studies were excluded because they already performed detailed characterization of plasmid gene expression of the plasmids pLM6179 and pLMR479a Cortes et al., 2020). In addition, another study (Kragh & Truelstrup Hansen, 2019) was excluded because no DE genes were detected on plasmid pLM5578. Two of the transcriptomes reanalyzed here for plasmid gene expression (Guariglia-Oropeza et al., 2018;Tang et al., 2015) utilized the L. monocytogenes strain H7858, a strain that was isolated from hot dogs during a listeriosis outbreak in 1998 (Centers for Disease Control and Prevention, 1998;Nelson et al., 2004). H7858 is a wellcharacterized L. monocytogenes strain and harbors the plasmid pLM80, which was assembled into two contigs (accession NZ_AADR01000010; NZ_AADR01000058). In these two studies, transcriptome reads were mapped to a pseudochromosome generated by aligning H7858 contigs to the chromosome of L. monocytogenes EGD-e. However, because EGD-e does not possess a plasmid, the H7858 plasmid contigs were not aligned and thus not incorporated into the H7858 pseudochromosome. As a result, any reads originating from pLM80 were not mapped and analyzed in either study.
In the first of these studies, Tang et al. (2015) compared the transcriptomes of H7858 grown at 7°C on vacuum-packed coldsmoked salmon (CSS) and in a modified brain heart infusion broth (MBHIB) (Tables 1 and 2). MBHIB is distinct from typical brain heart infusion broth (BHI) as the salt concentration was modified to 4.65%, and the pH was adjusted to 6.1 to better mimic conditions found on CSS. In the second study, Guariglia-Oropeza et al. (2018), cultured H7858 in BHI broth containing 1.1% porcine bile and compared gene expression against cells grown in standard BHI at pH 5.5 as a control (Tables 1 and 2). All incubations conducted by Guariglia-Oropeza et al. (2018) were performed at 37°C.
The final study chosen for this analysis was conducted by Assisi et al. (2021). In their investigation, the authors sequenced and assembled the genomes of 21 L. monocytogenes isolates derived from the deli retail environment, and four of these isolates were chosen for subsequent gene expression analysis comparing growth in biofilm versus planktonic broth growth (Tables 1 and 2). Similar to the other studies, the authors mapped reads to the genome of L. monocytogenes 10403S, a strain that lacks a plasmid, thus overlooking potential differences in plasmid gene expression between conditions.

| Data acquisition
Raw sequence reads from Tang et al. (2015) were obtained from the NCBI Sequence Read Archive database, and reads from Guariglia-Oropeza et al. (2018) and Assisi et al. (2021) were kindly provided by the authors. Detailed information about these data sets can be found in Table 1.

| Identification of putative plasmid contigs
Identification of putative plasmid contigs was done using the methodology described by Schmitz-Esser et al. (2021). Briefly, assembled L. monocytogenes genomes were first assessed for plasmid presence using L. monocytogenes RepA protein sequences as BLASTp queries. For this, the amino acid sequences representing a Group 2 RepA (pLM80, GenBank accession number WP_003726391) and a Group 1 RepA (pLM33, YP_003727990.1) were used as query sequences. Because L. monocytogenes plasmids are highly conserved T A B L E 1 Strains and sequencing information from published transcriptome sequencing data sets reanalyzed in this study for plasmid gene expression Tang et al. (2015) Guariglia
Putative ncRNAs were compared with and assessed for homologs in other bacteria using the nucleotide sequences as a query and searched against the RNA families (Rfam) database and server Abbreviations: BHI, brain heart infusion; CSS, cold-smoked salmon; MBHIB, modified brain heart infusion broth.
averaged in R to form consensus TPMs, and TPMs were ranked first (highest TPM value) to last (lowest TPM value) based on their average TPM value. Ranking of TPM expression level was used to qualitatively assess the transcriptional level of plasmid genes with respect to other genes on the same plasmid.

| RESULTS AND DISCUSSION
Our analysis found that two of the four isolates used for transcriptome sequencing in the study conducted by Assisi et al. (2021), FSL R8-5446 and FSL R8-7802, harbored a novel, putative plasmid: pLM5446 and pLM7802, respectively ( Table 3). The three plasmids in this study have been assembled in several contigs, with assembly sizes ranging from 73 to 135 kbp, and have a GC content ranging from 36.3% to 37.5% (Table 3). The aligned plasmid contigs showed high nucleotide sequence similarity between pLM80 and pLM7802 ( Figure 1). Notably, the entire pLM80 plasmid was contained within the pLM7802 contigs, and overlapping sequences were virtually identical with >99.9% nucleotide identities. The pLM80-like module of pLM7802 comprised 62% of the entire pLM7802 sequence. A low-to-moderate similarity was observed between pLM5446 and pLM80 and between pLM5446 and pLM7802 ( Figure 1, Appendix Table A1, and

| Genetic analysis of pLM80
L. monocytogenes H7858 harbors the well-studied plasmid pLM80, which is 82 kbp in size and possesses a Group 2 RepA protein (Locus_tag: LMOh7858_pLM80_0093) and several genes involved in stress response. These stress response genes include a cadmium resistance ATPase, cadA (Parsons et al., 2018), a benzalkonium chloride resistance cassette, bcrABC (Elhanafi et al., 2010), and the triphenylmethane reductase tmr which is known to increase tolerance toward crystal violet dye (Dutta et al., 2014). Additionally, pLM80 encodes a putative UV-damage repair protein, uvrX (Locus_ tag: LMOh7858_pLM80_0090), predicted to be a DNA polymerase that assists in the ultraviolet light stress-response system (Kuenne et al., 2010). Kuenne et al. (2010) suggested that the common presence of uvrX on L. monocytogenes plasmids may indicate a mechanism that produces genetic deletions to increase the variation of the plasmids during DNA repair. Kuenne et al. (2010) also identified a 40 kb region in pLM80 harboring a putative conjugation system containing predicted type IV secretion system proteins. pLM80 shares homology with the Bacillus anthracis plasmid pXO2, which shares a common backbone with the conjugative plasmid pAW63 of Bacillus thuringiensis (Van Der Auwera et al., 2005).
Two putative ncRNAs were predicted within pLM80 (Table 4), of which rli28 has previously been identified by Kuenne et al. (2010). In several L. monocytogenes strains, rli28 was also found on the chromosome where it is flanked by lmo0470 and lmo0471 homologs.
In addition, rli28 is a homolog (76% nucleotide identity and 86% query coverage) of the chromosomally encoded rli50 (also known as pig manure treatment processing unit), it was found that rli28 and rli50 were significantly downregulated in the lagoon effluent media (Vivant et al., 2017). In another study where rli50 was deleted, L. monocytogenes virulence was attenuated in a murine macrophage model (Mraheil et al., 2011).
The second pLM80 ncRNA predicted is ratA. This is the first time ratA is found within the sequences of an L. monocytogenes plasmid.
RatA is annotated as part of a TxpA/RatA type I toxin  is found immediately adjacent to the txpA/ratA toxin−antitoxin locus within the B. subtilis phage-like region known as the skin element (48 kb in size), and it is thought to be part of a type I toxin−antitoxin cassette similar to TxpA/RatA (Irnov et al., 2010;Silvaggi et al., 2005).
It is worth noting that BsrH was first identified as a lncRNA, and its role as a protein-encoding mRNA was only later elucidated (Irnov et al., 2010). The putative pLM80 bsrH gene overlaps rli28 by 105 bp on the same strand and extends 14 bp past the 3' end of rli28 ( Figure 2). It is tempting to speculate that Rli28 may be a cis-acting (ncRNAs that affect mRNA encoded in the same locus) signalresponse regulator for the putative toxin, as discussed briefly in other systems (Irnov et al., 2010), and that RatA may act as the antitoxin ncRNA for this system. However, this regulatory relationship needs to be experimentally verified in future studies.

| Genetic analysis of pLM7802
We found that pLM7802 was assembled into 12 contigs with an assembly size of 135 kbp and contained two RepA proteins, one from F I G U R E 2 Genetic organization and read mapping of the rli28, brsH, and ratA genes of pLM80, pLM5446, and pLM7802. The rli28, brsH, and ratA loci of pLM80, pLM5446, and pLM7802 are identical; thus, the locus from pLM80 was chosen as the representative for the figure of the overall genetic organization. The numerical track is delimited in 100 bp segments and represents the location of the genes within the pLM80 sequence. Dashed gray lines represent different reading frames of the strands with those that are above the numerical track representing the positive strand and those below indicating the negative strand. Transcriptome reads ( (Locus_tag: KJS00_15040) ( Table 3). The presence of two distinct RepA proteins may indicate that pLM7802 represents two plasmids, and indeed the phenomenon of L. monocytogenes strains harboring two plasmids has been reported before (Harrand et al., 2020;Hingston, Chen, Dhillon, et al., 2017;Kolstad et al., 1991;Korsak et al., 2019;Lebrun et al., 1992;Romanova et al., 2002). Alternatively, pLM7802 may comprise a single plasmid with two distinct repA Fluoride exhibits antimicrobial activity in water (Marquis, 1995); thus, increased tolerance to fluoride supplemented in tap water may enhance the stress survival of L. monocytogenes in food and FPEs.
Finally, four putative ncRNAs were predicted to be encoded on the pLM7802 plasmid contigs (Table 4): the aforementioned fluoride riboswitch, identical copies of the pLM80 ratA and rli28, and a putative NiCo riboswitch previously described in the L. monocytogenes plasmid pLMR479a (Cortes et al., 2020). Regarding the pLM7802 ratA, pLM7802 also harbors the same putative toxin gene identified on pLM80 (Locus tag: LMOh7858_pLM80_0050).

| Genetic analysis of pLM5446
pLM5446 was assembled into four contigs, has an assembly size of 73 kbp, and contains a Group 1 RepA protein gene (Locus_tag:

| General gene expression characteristics of L. monocytogenes plasmids
After quality filtering of the transcriptome reads and mapping against the respective L. monocytogenes genomes, plasmid coverages ranged ANAST ET AL.
| 7 of 17 from 21× to 420×, and total read-depth per sample (including chromosome and plasmid contigs) ranged from 1.6 to 38.8 million (Table 5 and

| pLM80 gene expression results
After L. monocytogenes strain H7858 was exposed to 1.1% porcine bile, the expression of 30 pLM80 genes (32% of total plasmid genes) significantly changed (  (1)  LMOh7858_pLM80_0005 and LMOh7858_pLM80_0049) that shows similarity to the Bacillus plasmids pXO2 and pWA63 (Korsak et al., 2019;Kuenne et al., 2010;Van Der Auwera et al., 2005). These data suggest that conjugation may occur on the surface of CSS.
Indeed, two of these genes are annotated as encoding elements of a The ability of pLM80 to transfer by conjugation is further supported by the fact that it is virtually identical to pLIS1, a plasmid from a Listeria welshimeri strain that showed high transformation efficiency from L. welshimeri to L. monocytogenes (Korsak et al., 2019). Overall, the data indicate that the conjugation of pLM80 during growth on CSS may occur.
In the same study, Tang et al. (2015) utilized the same batch of commercially produced wet-cured CSS fillets as Kang et al. (2012) used to characterize the composition and physicochemical characteristics of the naturally occurring commensal bacteria found on the CSS fillets. Most of the CSS microbiota was comprised of lactic acid bacteria (LAB) found at concentrations as high as 7 log CFU/g (Kang et al., 2012). It has been established that LAB compete against and even inhibit L. monocytogenes proliferation and survival (Gómez et al., 2016;Lewus et al., 1991;Scatassa et al., 2017). Therefore, Assessing pLM80 gene expression levels from Tang et al. (2015) samples revealed that the most expressed genes in the control (MBHIB) and experimental (CSS) replicates were within the same region that includes the bcrABC cassette, tmr, a gene encoding a putative glyoxalase family protein, and two putative resolvases (Table S3: https://doi.org/10.6084/m9.figshare.20558514).
Lastly, 13 pLM80 genes were DE in both studies included here (Guariglia-Oropeza et al., 2018;Tang et al., 2015) and are listed in Table 6 with their corresponding log 2 FCs. Except for one hypothetical protein, all genes that were DE in both studies showed opposite changes in expression in the two studies. Together with the differences in gene expression described above, this suggests that the pLM80 gene expression patterns are specific to the fundamentally different conditions applied in the two studies.

| pLM7802 gene expression results
Analyzing the plasmid transcriptome of L. monocytogenes FSL R8-7802 planktonically cultured in BHI broth compared to cells adhered to steel coupons revealed that 33 pLM7802 genes were DE (21% of genes on the plasmid). All DE genes were significantly upregulated with log 2 FCs ranging from 1. L. monocytogenes uvrX-like plasmid genes have been DE during cocultivation with cheese bacteria, salt stress, and organic and inorganic acid stress Cortes et al., 2020;Hingston et al., 2019). Interestingly, putative uvrX genes were consistently DE in various L. monocytogenes stress exposure studies and during planktonic versus biofilm growth comparisons. These observations suggest that the putative uvrX gene has one or more functions advantageous for L. monocytogenes strains.

| pLM5446 gene expression results
Comparing planktonic growth in BHI broth against L. monocytogenes FSL R8-5446 in biofilm on stainless steel coupons revealed significant upregulation of 40 pLM5446 genes (51% of total plasmid genes) and FC 4.0); homologs of this system found on the plasmid pLM6179 were upregulated during coculture with cheese bacteria and exposure to lactic acid stress Cortes et al., 2020). As mentioned previously, pLM5446 encodes four putative genes involved in heavy metal resistance. All four genes were upregulated with log 2 FCs ranging from 1.3 to 3.5 (Locus_tags: KJR89_15405, KJR89_15440, KJR89_15110, and KJR89_15445).
One possible explanation for the significant upregulation of putative heavy metal tolerance genes is that cells adhered to stainless steel plates (ASI 304) in biofilm experiments. ASI 304 stainless steel contains iron, nickel, and chromium, which may leach from the coupons and come in contact with L. monocytogenes (Jellesen et al., 2006). In addition, other putative heavy metal tolerance genes were upregulated as well: cadA2 (Locus_tag: KJR89_14955, log 2 FC 2.6), cadC2 (Locus tag: KJR89_14960, log 2 FC 1.1), and cadD (Locus tag: KJR89_15115, log 2 FC 0.8).
Other genes of interest that were upregulated included uvrX (Locus tag: KJR89_15035-log 2 FC 3.5), and, similar to pLM7802, the gene encoding the putative NADH peroxidase (Locus_tag: KJR89_15095) was upregulated in biofilm with a log 2 FC of 1.2.
Notably, the most upregulated genes were the two copies of rli28 Comparing the differential gene expression of the pLM7802 and pLM5446 under the same conditions revealed that 11 shared genes were DE in both plasmids during growth in biofilm (Table 7).
However, the majority of DE genes for each of the plasmids were plasmid-specific. Interestingly, all shared DE genes were consistently upregulated during growth in biofilm. It is currently unknown which of these shared plasmid genes are involved in biofilm formation or if unique genes on individual plasmids might be involved in biofilm formation. Future studies will be needed to determine if any plasmid genes are involved in biofilm formation.

| Plasmid-encoded ncRNAs exhibit high levels of transcription
Previous transcriptomic analysis of L. monocytogenes revealed notably high expression of chromosomal Cortes et al., 2020;Duru et al., 2021) and plasmid (Cortes et al., 2020) ncRNAs compared to the expression of other genes in the same conditions. Thus, in addition to elucidating differential expression of protein-coding genes harbored on pLM80, pLM5446, and pLM7802, we sought also to determine if the putative ncRNAs described in this study were highly expressed relative to chromosomal and plasmid-encoded protein-coding genes (Table S3: https://doi.org/10.6084/m9.figshare.20558514). Remarkably, rli28 and the putative NiCo riboswitch were among the five most expressed plasmid genes in the experimental and control replicates of most data sets described here, with the notable exception of rli28 showing slightly lower TPM rankings of 9th and 11th in the replicates of Tang et al. (2015). The high expression levels of these ncRNAs indicate that plasmid ncRNAs may have a role in plasmid gene regulation and potentially regulate plasmid stress tolerance mechanisms. However, the function of these putative plasmid ncRNAs will need to be determined experimentally in future studies.

| CONCLUSION
Until now, only three studies have analyzed the complete plasmid transcriptome profiles of L. monocytogenes strains Cortes et al., 2020;Kragh & Truelstrup Hansen, 2019).
In the data sets reanalyzed here, we observed that many plasmid genes were DE (21%−51% of the total plasmid genes). Furthermore, a number of the aforementioned DE genes were also DE in previous analyses Cortes et al., 2020). Although most DE plasmid genes from this study possess no predicted function, the substantial number of DE genes in each data set suggests that plasmid genes have an important-yet unknown-role in the L. monocytogenes response to the tested conditions. Therefore, we emphasize the need for further analysis of these genes and their encoding plasmids to determine their functions and potential roles in the survival and persistence of L. monocytogenes.
Notably, even though L. monocytogenes strains FSL R8-7802 and FSL R8-5446 were grown under the same conditions (biofilm and planktonic growth), the gene expression patterns of their plasmids, pLM7802 and pLM5446, were highly distinct. These data suggest that even if plasmids harbor several genes encoding near-identical proteins, gene expression seems to be plasmid-specific even under the same experimental conditions. The difference in the expression of similar plasmid genes encoded on different plasmids may be due to divergent gene regulatory systems, plasmid-or chromosomally encoded. Plasmid−chromosomal cross-talk of gene regulation systems does occur in other bacteria (Diel et al., 2019;Gong et al., 2013;Vial & Hommais, 2020) and may occur in L. monocytogenes as well.
We identified putative ncRNAs that were DE in multiple highly distinct conditions on every plasmid analyzed. Furthermore, rli28 and Stephan Schmitz-Esser: Conceptualization (equal); funding acquisition (lead); supervision (lead); writing-original draft (equal); writing-review and editing (equal). pLM5446, pLM7802, and pLM80 genome information and gene expression results from the studies by Assisi et al., 2021, Guariglia-Oropeza et al., 2018, and Tang et al., 2015.) This is then followed by comparative protein blasts of pLM7802 against pLM80, pLM7802 against pLM5446, and pLM80 against pLM5446. The similarity between protein sequences is denoted as the percent amino acid identity shared between the plasmids and then by percent query coverage. Gray rows indicate that the corresponding CDS was duplicated within the plasmid module to align homologs found between each plasmid. This was done to account for several predicted protein sequences from pLM80 and pLM5446 that had multiple homologs within the pLM7802 proteome. Within the individual plasmid modules, "Locus tag" indicates the assigned locus tag of each predicted CDS, "Product" denotes the predicted final product from each gene, "Gene name" designates the short name of each gene where applicable, and "Pfam protein domain(s)" shows the predicted Pfam protein domains within each amino acid sequence generated by the eggNOG-mapper (http://eggnog-mapper.embl.de/).

ACKNOWLEDGMENTS
Gene expression results are first assigned by the publication, followed by either "log2FoldChange" (log 2 fold change of gene) or "Q-value" (p value adjusted for errors from multiple testing). Gene expression data are only displayed for genes that had Q values ≤ 0.05;  were submitted to the SRA are not the raw reads containing pLM80 reads. We obtained unfiltered reads by directly contacting the authors of this study. "Plasmid" denotes what plasmid is harbored within the strain from the sample. "Total mappings" means the total reads that were mapped to the chromosome and plasmid contigs.
"Length of the entire genome (sum contigs)" shows the sum of all base pairs from the chromosome and plasmid contigs. "Length of plasmid (sum contigs)" designates the sum of base pairs of the predicted plasmid contigs. "Reads mapped to plasmid" displays the total reads mapped to plasmid-only contigs. "Chromosome coverage" denotes the total read coverage of all the chromosome-only contigs.
"Plasmid coverage" indicates the total read coverage of all plasmidonly contigs. "Percent of Total mappings that are attributed to plasmid contigs" shows the percentage of all mapped reads in the sample that were assigned to plasmid contigs; Table S3 is available at https://doi.org/10.6084/m9.figshare.20558514 (Table S3. Normalized gene expression levels of genes from the plasmids pLM7802, pLM5446, and pLM80.) Results are separated by plasmids in sheets named after the plasmids pLM7802, pLM5446, and pLM80. At the highest level, results are presented by study, then by experimental condition, and lastly, by expression results and locus tags or ncRNA name. "Locus tags/ncRNA name" indicates the assigned locus tag of each predicted CDS or the name of a predicted ncRNA. "TPM" denotes the transcript per million value for each gene generated by ReadXplorer and with TPM values of each replicate averaged together in R. "Rank" shows the numerical order from greatest TPM value to smallest TPM value starting with the greatest value as "1" relative to the plasmid genes of each experimental condition. A TPM value of "-" indicates that no reads were mapped for that specific gene in the corresponding data set).

ETHICS STATEMENT
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APPENDIX A
See Figure A1 and Table A1.
F I G U R E A1 Amino acid alignment of putative multicopper oxidases from Listeria monocytogenes and multicopper oxidases of Staphylococcus aureus and Escherichia coli. The alignment was conducted with MAFFT (https://mafft.cbrc.jp/alignment/server), and conserved amino acid residue shading was done with Boxshade. Asterisks show identical positions, and dots highlight similar positions (threshold fraction 0.7). Amino acid residues important for function in the characterized CueO from E. coli are highlighted in pink and green (X. Li et al., 2007) and pink (Kataoka et al., 2009).